Succinate dehydrogenase is an indispensable enzyme involved in the Krebs cycle as well as energy coupling in the mitochondria and certain prokaryotes. During catalysis, succinate oxidation is coupled to ubiquinone reduction by an electron transfer relay comprising a flavin adenine dinucleotide cofactor, three iron-sulfur clusters, and possibly a heme b 556 . At the heart of the electron transport chain is a [4Fe-4S] cluster with a low midpoint potential that acts as an energy barrier against electron transfer. Hydrophobic residues around the [4Fe-4S] cluster were mutated to determine their effects on the midpoint potential of the cluster as well as electron transfer rates. SdhB-I150E and SdhB-I150H mutants lowered the midpoint potential of this cluster; surprisingly, the His variant had a lower midpoint potential than the Glu mutant. Mutation of SdhB-Leu-220 to Ser did not alter the redox behavior of the cluster but instead lowered the midpoint potential of the [3Fe-4S] cluster. To correlate the midpoint potential changes in these mutants to enzyme function, we monitored aerobic growth in succinate minimal medium, anaerobic growth in glycerol-fumarate minimal medium, non-physiological and physiological enzyme activities, and heme reduction. It was discovered that a decrease in midpoint potential of either the [4Fe-4S] cluster or the [3Fe-4S] cluster is accompanied by a decrease in the rate of enzyme turnover. We hypothesize that this occurs because the midpoint potentials of the [Fe-S] clusters in the native enzyme are poised such that direction of electron transfer from succinate to ubiquinone is favored. Electron transport (ET)3 chains are ubiquitous and play a key role in energy conservation in both aerobic and anaerobic respiration. Cofactors such as iron-sulfur ([Fe-S]) clusters, hemes, and flavins comprise the ET relays of respiratory chain enzymes and mediate electron transfer from a powerful reductant with a relatively low midpoint potential (E m ) to a final oxidant with a relatively high E m . The ET chain usually involves cofactors from multiple enzymes and the membrane-soluble ubiquinone or menaquinone pool, and the energetics of individual ET steps are not always downhill. In many redox enzymes, the exception often occurs in the form of an [Fe-S] cluster with an unusually low E m located at an intermediate position in the ET relay (1-4). Thus during catalysis, electrons must surmount the energy barrier imposed by the low potential cluster despite the overall downhill reaction between the reductant and oxidant. Controversy continues to surround the issue as to whether E m values of cofactors play a role in determining the rate of electron transfer through redox enzymes, especially that of the low potential cluster (5-10). It would thus be of great interest to use a genetically modifiable model system to study the effects of E m modulation on observed catalytic rates of electron transfer.Succinate dehydrogenase (Sdh, Complex II in eukaryotes) is an indispensable enzyme involved in the Krebs cycle and...

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